The present invention relates to a library of catalysts of interest coupled to a substrate by an exchangeable linker pair, X and Y, and a selection method that uses multiple catalytic turnover events to isolate the more active of the catalysts in said library.
In the past, novel biopolymer (i.e. DNA, RNA or polypeptide) based catalysts have been created in several different ways. The following paragraphs describe some of these selection schemes.
Catalytic RNA, DNA and protein (particularly antibodies) have been isolated by this approach. It has mostly been applied to the isolation of catalytic antibodies by immunization of mice with transition state analogs (TSA), also antibodies displayed on phage as well as RNA and DNA libraries have been challenged with TSA. The idea is that a molecule (protein, RNA or DNA) that binds a given TSA is likely to bind the substrate and stabilize the geometry and/or energetics of the transition state. This may result in catalysis.
The method does not select for catalytic activity per se, but rather for binding to a transition state analog (TSA). However, it has been included here as it is currently one of the most used methods to isolate novel catalysts. Problems encountered with this approach include: i) Detailed mechanistic knowledge of the target reaction is required (in order to design an appropriate TSA); ii) In many cases a TSA that adequately resembles the transition state is unobtainable or unstable; iii) It is not possible to mimic the structural and electronic dynamics of the reaction coordinate.
Consequently, a rather limited set of reaction types have been successfully targeted by this approach. In most cases the isolated catalysts have poor turn-over numbers.
This selection scheme has been applied to protein and nucleic acids. The substrate is designed so that a reactive product is formed during the reaction (the substrate is called xe2x80x9csuicide substratexe2x80x9d or xe2x80x9cinhibitor analogxe2x80x9d). The reactive product is likely to react with the catalyst that produced it, to form a covalent bond. As a result, active catalysts can be separated from inactive ones by way of the attached label. Catalytic antibodies displayed on phage have been isolated by this method, and it was shown in a model system that catalytically active and inactive proteins could be separated using this approach. The method should allow the isolation of rare catalysts.
Important limitations with this approach include: i) For many reactions it is not possible to design an appropriate suicide substrate. ii) Successful catalysts need only perform one turn-over during the selective process/round, which is typically on the order of minutes. Hence, there is no selective advantage for efficient catalysts.
RNA libraries have been designed that contain both the substrate and the potentially catalytic domain in the same molecule. RNAs capable of performing the desired reaction (typically ligation) will xe2x80x9cactivatexe2x80x9d themselves for amplification (reverse transcription followed by RNA polymerase transcription). By adequate dilutions and additions of nucleotide precursors this continuous selection can be maintained over several hours, and then analyzed.
The method has two important limitations: i) Both the substrate and the catalyst must be a nucleic acid; ii) As the catalyzed reaction and the amplification of successful enzymes is not separated, the time of the selective step is the sum of the turn-over time of the target reaction and the time of amplification of the xe2x80x9cactivatedxe2x80x9d molecules. Thus, as the amplification is on the order of seconds, there is no selective advantage for an efficient catalyst.
Recently, methods have been described, involving the attachment of the substrate of the target reaction to a protein with potential catalytic activity towards the attached substrate (Pedersen et al., Proc. Natl. Acad. Sci., US, 1998, vol. 95, pp. 10523-10528; Jestin et al., 1999, Angew. Chem. Int. Ed., vol. 38, pp. 1124-1127; Demartis et al., 1999, JMB, vol. 286, pp. 617-633; Neri et al., 1997, WO 97/40141). Upon intramolecular conversion of the substrate, the active catalyst can be isolated by means of the attached product.
This scheme is very general. However, since successful catalysts need only perform one turn-over during the selective process/round, which is typically in the order of minutes, there is no selective advantage for efficient catalysts. For the same reason, it presumably is not possible to distinguish enzymes with slightly different specific activity with this selection scheme.
The problem to be solved by the present invention is to provide a method for in vitro selection, from a library of catalyst molecules, of a catalyst molecule of interest having a relatively more efficient specific catalytic activity of interest, as compared to the rest of the catalyst molecules within said library, and wherein said in vitro selection method is characterised by that it allows multiple catalytic activity turn-overs (i.e. substrate to product catalytic activity turn-overs), by the catalyst molecule of interest, before it is finally collected.
The solution is based on using a novel sample comprising a number of individual units in said in vitro selection method.
A summary of the characteristics of said novel sample is given immediately below.
Said novel sample comprises a library of catalyst molecules provided in the form of individual units, wherein the individual units comprise a first type individual unit having the following general structure:
Cxe2x80x94XYxe2x80x94S,
wherein C denotes a catalyst molecule, XY an XY exchange pair, and S a substrate which is capable of being catalysed into a product by at least one catalyst comprised within said library of catalyst molecules and thereby providing the possibility of obtaining a second type individual unit comprising the general structure:
Cxe2x80x94XYxe2x80x94P,
wherein C and XY has the meaning defined above and P is the product molecule resulting from the catalytic conversion of the substrate S of the first type individual unit. See FIG. 1 for a graphic illustration of a suitable example of such an individual unit.
Said novel sample, is then characterised by that it comprises following functionally defined features:
Feature 1:
The substrate S is attached to the catalyst in a configuration that allows catalytic reaction between the catalyst and the substrate within said individual unit; and
the nature of said attachment of the substrate and the catalyst provides the possibility, by means of a characteristic of the product, of isolating an entity comprising information allowing the unambiguous identification of the catalyst molecule which has been capable of catalysing the reaction substrate molecule to product molecule.
For illustration reference is made to FIG. 1, where is shown a suitable example of such an individual unit comprising feature 1 above.
Feature 2:
Said sample comprising a number of individual units and comprising feature 1 above is further characterised by that said XY exchange pair allows an asymmetric exchange of the Y-moiety with another Y-moiety (i.e. Y exchanges with Y, not with X); whereby
said XY exchange pair then allows an exchange reaction between the unit structure:
a catalystxe2x80x94an XY exchange pairxe2x80x94a product and a xe2x80x9cYxe2x80x94substratexe2x80x9d component
and thereby generating the unit structure
a catalystxe2x80x94an XY exchange pairxe2x80x94a substrate.
For illustration reference is made to FIG. 2, where is shown a suitable example of such an individual unit comprising feature 2 above.
Using such a novel sample in a in vitro selection method as described herein (vide infra), provides then the possibility of selecting a catalyst molecule of interest essentially only based on a characteristic of the product molecule which has been generated by the catalyst molecule of interest (Feature 1 allows this; see FIG. 1 for an illustration);
and it further provides the possibility of selecting a catalyst molecule of interest having a relatively more efficient specific catalytic activity of interest, as compared to the rest of the catalyst molecules within said library, and wherein said selection is characterised by that said catalyst molecule of interest is first finally collected after it has been allowed to perform multiple catalytic activity turn-overs (i.e. substrate to product catalytic activity turn-overs) (Feature 2 allows this; see FIG. 2 for an illustration).
Accordingly, a first aspect of the invention relates to a sample comprising a number of individual units suitable for use in an in vitro selection system, wherein the purpose of said in vitro selection system is, from a library of catalyst molecules, to select a catalyst molecule of interest having a relatively more efficient specific catalytic activity of interest as compared to the rest of the catalyst molecules within said library, and wherein said in vitro selection system is characterised by that it allows multiple catalytic activity turn-overs (i.e. substrate to product catalytic activity turn-overs), by the catalyst molecule of interest, before it is finally collected and wherein said sample comprises,
(i) a library of catalyst molecules provided in the form of individual units, wherein the individual units comprise a first type individual unit having the following general structure:
Cxe2x80x94XYxe2x80x94S,
wherein C denotes a catalyst molecule, XY an XY exchange pair, and S a substrate which is capable of being catalysed into a product by at least one catalyst comprised within said library of catalyst molecules and thereby providing the possibility of obtaining a second type individual unit comprising the general structure:
Cxe2x80x94XYxe2x80x94P,
wherein C and XY has the meaning defined above and P is the product molecule resulting from the catalytic conversion of the substrate S of the first type individual unit; and
(a) the substrate S is attached to the catalyst in a configuration that allows catalytic reaction between the catalyst and the substrate within said individual unit; and
(b) the nature of said attachment of the substrate and the catalyst provides the possibility, by means of a characteristic of the product, of isolating an entity comprising information allowing the unambiguous identification of the catalyst molecule which has been capable of catalysing the reaction substrate molecule to product molecule; and
(c) said sample comprising a number of individual units is characterised by that said XY exchange pair allows an asymmetric exchange of the Y-moiety with another Y-moiety (i.e. Y exchanges with Y, not with X); whereby said XY exchange pair then allows an exchange reaction between the unit structure:
a catalystxe2x80x94an XY exchange pairxe2x80x94a product
and a xe2x80x9cYxe2x80x94substratexe2x80x9d component
and thereby generating the unit structure
a catalystxe2x80x94an XY exchange pairxe2x80x94a substrate.
The term xe2x80x9can individual unitxe2x80x9d, comprised within a sample according to the first aspect as described above, denotes an individual unit comprising the general structure as specified under point (i) in the first aspect of the invention; a substrate molecule attached to a catalyst molecule as specified under point (a) and (b) in the first aspect of the invention; and an XY exchange pair as specified under point (c) in the first aspect of the invention. See FIGS. 1 and 2 for a suitable example of such an individual unit.
The term xe2x80x9can individual unit comprising the general structure: a catalystxe2x80x94an XY exchange pairxe2x80x94a substrate; or a catalystxe2x80x94an XY exchange pairxe2x80x94a productxe2x80x9d denotes that said individual unit comprises at least one molecule of each of said entities, i.e. at least one catalyst molecule, at least one XY exchange pair molecule, and at least one substrate molecule or at least one product molecule. Accordingly, said individual unit may for instance comprise more than one copy of an identical catalyst molecule or may comprise several different catalyst molecules.
Further the term xe2x80x9cxe2x80x94xe2x80x9d placed between the individual entities within said individual unit denotes that there is a physical connection between said individual entities within said individual unit, i.e. that there is a physical connection between a catalystxe2x80x94an XY exchange pairxe2x80x94a substrate or
a catalystxe2x80x94an XY exchange pairxe2x80x94a product.
Further, xe2x80x9can individual unitxe2x80x9d as described herein denotes an individual unit wherein it is possible to physically separate said individual unit from the other different individual units, within said sample, in order to be able to isolate the separate individual unit.
The term xe2x80x9cdifferent individual unitsxe2x80x9d denotes different individual units each independently comprising different catalyst molecules, i.e. an example of two different individual units may be
(1) catalyst molecule1xe2x80x94XY exchange pairxe2x80x94substrate; and
(2) catalyst molecule2xe2x80x94XY exchange pairxe2x80x94substrate;
wherein catalyst molecule1 and catalyst molecule2 denotes two different catalyst molecules.
The term xe2x80x9ca sample comprising a number of different individual unitsxe2x80x9d denotes a sample comprising at least two different individual units, preferably at least 100 different individual units, more preferably at least 10.000 different individual units, more preferably at least 106 different individual units, more preferably at least 108 different individual units, more preferably at least 1010 different individual units, even more preferably at least 1012 different individual units, and most preferably at least 1014 different individual units. Basically the actual number of different individual units corresponds to the actual size of the library of catalyst molecules.
The term xe2x80x9ca sample comprising a number of individual unitsxe2x80x9d and the term xe2x80x9ca sample comprising a number of different individual unitsxe2x80x9d may be used interchangeably herein.
The term xe2x80x9ca library of catalyst moleculesxe2x80x9d denotes a library comprising at least two different catalyst molecules, preferably at least 100 different catalyst molecules, more preferably at least 10.000 different catalyst molecules, more preferably at least 106 different catalyst molecules, more preferably at least 108 different catalyst molecules, more preferably at least 1010 different catalyst molecules, even more preferably at least 1012 different catalyst molecules, and most preferably at least 1014 different catalyst molecules.
The term xe2x80x9ca substrate capable of being catalysed into a product molecule by at least one catalyst molecule comprised within said library of catalyst moleculesxe2x80x9d basically denotes any suitable substrate molecule. Essentially said substrate molecule is chosen according to the specific catalytic activity which it is desired to select for. For instance, if the desired catalytic activity is a protease activity then a suitable substrate may be a peptide molecule and the product will then be a degraded peptide. The terms xe2x80x9csubstratexe2x80x9d and xe2x80x9csubstrate moleculexe2x80x9d may be used interchangeably.
The term xe2x80x9cproductxe2x80x9d denotes the product obtained by the catalytic reaction substrate to product by a catalyst of interest as specified herein. The terms xe2x80x9cproductxe2x80x9d and xe2x80x9cproduct moleculexe2x80x9d may be used interchangeably.
The term xe2x80x9ccatalystxe2x80x9d denotes any catalyst molecule with a desired catalytic activity, such as organic and inorganic molecules, proteins, enzymes, peptides, nucleic acids, biopolymers and non-biological polymers, small organic or inorganic molecules. The terms xe2x80x9ccatalystxe2x80x9d and xe2x80x9ccatalyst moleculexe2x80x9d may be used interchangeably.
The term xe2x80x9cthe substrate is attached to the catalyst in a configuration that allows catalytic reaction between the catalyst and the substrate within said individual unitxe2x80x9d denotes a direct or indirect physical connection, within each of the individual units, between substrate and catalyst. This connection should preferably maximize productive interaction of the catalyst and the substrate, within the individual unit, while minimizing the interaction of catalysts and substrates on different individual units.
The term xe2x80x9cthe nature of said attachment of the substrate and the catalyst provides the possibility, by means of a characteristic of the product, of isolating an entity comprising information allowing the unambiguous identification of the catalyst molecule which has been capable of catalysing multiple times the reaction substrate molecule to product moleculexe2x80x9d according to point (b) of the first aspect of the invention denotes that said entity is isolated by means of one or more characteristic of the product.
An example of a suitable characteristic of the product may be that said product does not bind to a matrix and the substrate does bind to a matrix. In this case a suitable selection protocol may be that the individual units are bound to the solid support on the form a catalystxe2x80x94an XY exchange pairxe2x80x94a substratexe2x80x94matrix, and released when it is on the form catalystxe2x80x94an XY exchange pairxe2x80x94a product. For a detailed description of an example of such a system reference is made to a working example herein (vide infra).
Another example of a suitable characteristic of the product may be that said product binds to a receptor as illustrated in FIG. 1.
The term xe2x80x9can entity comprising information allowing the unambiguous identification of the catalyst molecule which has been capable of catalysing the reaction substrate molecule to product moleculexe2x80x9d according to point (b) in the first aspect of the invention, denotes either an entity wherein said information is carried in the catalyst molecule as such or an entity comprising other kind of information providing the possibility of unambiguously identifying the catalyst. Such other kind off information may for instance be an entity comprising a DNA sequence encoding a peptide or a polypeptide when the catalyst molecule of interest is a peptide or a polypeptide. An illustration of this may be when the isolated entity is a filamentous phage comprising a DNA sequence encoding a polypeptide of interest attached on the surface of said phage. See e.g. FIG. 12 and below for further details.
The term xe2x80x9cXY exchange pairxe2x80x9d, comprised within an individual unit, as specified above, denotes that the catalyst is attached to a substrate through an XY exchange moiety, i.e., the individual unit has the following general structure: a catalystxe2x80x94an XY exchange pairxe2x80x94a substrate and said XY exchange pair fulfils the criteria according to point (c) in the first aspect of the invention.
Preferably, the XY moiety is stable in the absence of free Y, but allows fast and specific exchange of free Y with Y bound to X (but not exchange of free Y with X). This exchange reaction can replace product with substrate, if the individual unit: a catalystxe2x80x94an XY exchange pairxe2x80x94a product is in contact with a Y-Substrate compound.
The XY exchange pair may for instance have following characteristics:
i) X and Y can be covalently or non-covalently bonded; and
ii) the XY exchange pair may consist of any kind of molecules, including small organic (eg., EDTA) and inorganic (eg. metals, phosphates) molecules as well as macromolecules (eg., nucleic acids, peptides).
See below for further details and FIGS. 2, 3 and 4 for graphic illustrations.
In a second aspect the invention relates to a method for in vitro selection, from a library of catalyst molecules, of a catalyst molecule of interest having a relatively more efficient specific catalytic activity of interest as compared to the rest of the catalyst molecules within said library and wherein said in vitro selection method is characterised by that it allows multiple catalytic activity turn-overs (i.e. substrate to product catalytic activity turn-overs), by the catalyst molecule of interest, before it is finally collected and wherein said method comprises following steps,
(i) placing a sample comprising a number of individual units according to the invention under suitable conditions where a catalyst molecule of interest performs its catalytic activity of interest and further under conditions wherein said individual units are in contact with an Yxe2x80x94substrate compound;
(ii) selecting for a catalyst of interest by selecting for one or more individual unit(s) which comprise(s) the product molecule; and
(iii) isolating an entity comprising information allowing the unambiguous identification of the catalyst molecule of interest which has been capable of catalysing multiple times the reaction substrate to product, by means of a characteristic of the product; and optionally
(iv) repeating step (i) to (iii) one or more times by using the information comprised in said entity of step (iii) to generate the catalyst molecule of interest and construct an individual unit comprising said generated catalyst molecule of interest and then using this individual unit as a starting material in said repetition step.
The term xe2x80x9cunder suitable conditions where a catalyst molecule of interest performs its catalytic activity of interestxe2x80x9d according to step (i) of the second aspect of the invention, denotes any suitable conditions where a catalyst molecule of interest performs its catalytic activity of interest.
Such suitable conditions may be alkaline pH if the purpose of the selection is to identify a catalyst of interest having activity at alkaline pH.
The term xe2x80x9csaid individual units are in contact with an Yxe2x80x94substrate compoundxe2x80x9d according to point (i) in second aspect of the invention denotes that the individual units and the Yxe2x80x94substrate compound are appropriately close that an exchange reaction, as specified under point (c) in the first aspect of the invention, is possible. Said contact may for instance be in a buffer solution wherein the individual units and the Yxe2x80x94substrate compound may diffuse together and thereby get in contact with each other. See FIG. 2 for graphic illustrations.
The term xe2x80x9cthe catalyst molecule of interest which has been capable of catalysing multiple times the reaction substrate to productxe2x80x9d according to step (iii) of the second aspect of the invention denotes that said catalyst molecule of interest has performed the catalytic reaction substrate to product at least two times, more preferably at least 100 times, more preferably at least 10.000 times, even more preferably at least 106 times, and most preferably at least 1010 times.
The term xe2x80x9can entity comprising information allowing the unambiguous identification of the catalyst molecule of interestxe2x80x9d denotes either an entity wherein said information is carried in the catalyst molecule as such or an entity comprising other kind of information providing the possibility of unambiguously identifying the catalyst. Such other kind of information may for instance be an entity comprising a DNA sequence when the catalyst molecule of interest is a peptide or a polypeptide. This is the same definition as described above for the same term in relation to point (b) of the first aspect of the invention (vide supra).
The term xe2x80x9crepeating step (i) to (iii) one or more times by using the information comprised in said entity of step (iii) to generate the catalyst molecule of interest and construct an individual unit comprising said generated catalyst molecule of interest and then using this individual unit as a starting material in said repetition stepxe2x80x9d according to point (iv) in the second aspect of the invention denotes that said repetition may be one time, more preferably 2 times, more preferably more than 5 times, even more preferably more than 10 times, and most preferably more than 25 times.
An advantage of the method for in vitro selection as described above is that it allows the catalyst molecules to perform multiple turn-overs of substrate to product during one selection round (i.e. before the catalyst molecule(s) of interest is finally collected).
This is fundamentally different from selection protocols previously described in the art, which either involved binding to a transition state analog of the target reaction, wherein there is no turn-over of substrate (see #1 in xe2x80x9cBackgroundxe2x80x9d above) or a single turn-over of substrate (#2 and 3 in xe2x80x9cBackgroundxe2x80x9d above).
This may provide two advantages, which may be illustrated by following a possible selection scheme using the method of the invention:
a) placing, according to point (i) of the method of second aspect of the invention, the sample comprising a number of individual units under suitable conditions where a catalyst molecule of interest exhibits its catalytic activity of interest and further under conditions wherein said individual units are in contact with an Yxe2x80x94substrate molecule at one end (starting end) of a product-binding column wherein a receptor specifically binding the product is coupled to the matrix of the product-binding column and with a suitable amount of the Yxe2x80x94substrate within the product-column buffer;
b) selecting, according to point (ii) of the method of second aspect of the invention, for a catalyst molecule of interest by selecting for one or more individual unit(s) which comprise(s) the product molecule, by collecting the individual unit(s) which arrive(s) latest to the opposite end (collecting end) of the column.
Within this selection scheme the following events, among others, take place within the product-column:
1) individual units comprising a potential catalyst molecule of interest converts the attached substrate to product;
2) said individual units, now comprising the product, are diffusing to and binding to a receptor placed close to the starting end of the product column;
3) the Yxe2x80x94substrate molecules, within the product-column buffer mediates the exchange reaction catalyst moleculexe2x80x94XY exchange pairxe2x80x94product and a Yxe2x80x94substrate molecule and thereby generating the unit structure catalyst moleculexe2x80x94XY exchange pairxe2x80x94substrate, and thereby mediates release of said bound individual units of step (2) above;
4) said released individual units of step (3) above are now comprising a substrate and a potential catalyst molecule of interests, which has made one catalytic conversion of the substrate to product, and is now regenerated in the original form of the individual units of point 1. Accordingly, said units can therefore once more perform reaction (1); binding to a receptor relatively closer to the collecting end of the product column etc.
The catalyst molecule of interest having most efficient specific catalytic activity of interest will perform most substrate to product conversions in a given time interval, and as a result, will spend more time immobilised on the product-binding receptor. Therefore, the individual unit(s) comprising said most efficient catalyst molecule will arrive latest to the collecting end of said column.
Using this example of a method of the second aspect of the invention, two advantages over the art may be:
i) that the possible xe2x80x9cselectablexe2x80x9d catalytic activity may be much higher than for the prior art selection protocols. A selective step involves performing the target reaction, diffusion to and binding of product to product-binding column, and finally exchange of product and substrate by the XY-exchange reaction. Since diffusion is generally a fast process, the selection stringency can be controlled simply by the amount of product binding receptor on the column or the type/amount of Yxe2x80x94S component in the product-column buffer; moreover, if for example electrophoresis is used as the means of isolating the more active catalysts, diffusion to the receptor may not be a limiting factor, wherefore the selection stringency may be even higher with this system;
ii) minor differences in activity can be distinguished; Since the selective step is reiterated many times as the catalysts flow through the column, even minor differences in catalyst activity will result in differential retention on the column, and thus differential enrichments.
In a final aspect the invention relates to a method for producing a catalyst molecule of interest comprising performing the method for in vitro selection according to the invention and the further following step,
(a) producing said isolated catalyst molecule of interest in a suitable quantity of interest by a suitable production method.